Calcium balance in sea bream (Sparus aurata): the effect of oestradiol-17beta

In all teleost fishes vitellogenesis is triggered and maintained by oestradiol-17beta (E2) and is accompanied by an increase of blood plasma calcium and phosphate. The action of this hormone on calcium metabolism was investigated by treating fast-growing immature juvenile sea bream (Sparus aurata) with coconut butter implants alone (control) or implants containing 10 microg/g E2. Treatment with E2 induced the production of circulating vitellogenin, a 2.5-fold increase in plasma ionic Ca2+ and a 10-fold increase in plasma total calcium, largely bound to protein. In contrast to freshwater species, which obtain most of their calcium from the environment directly through the gills, the intestinal component of calcium uptake of the salt water-living sea bream represented up to 60-70% of the total uptake. The whole body calcium uptake, expressed as the sum of calcium obtained via intestinal and extra-intestinal (likely branchial) routes increased significantly in response to E2. Combined influx and unchanged efflux rates resulted in a significant 31% increase in net calcium uptake. There was no evidence for an effect of E2 on the calcium and phosphate content of the scales or the tartrate-resistant acid phosphatase activity (an index for bone/scale osteoclast activity). While most freshwater fish appear to rely on internal stores of calcium, i.e. bone and/or scales to increase calcium availability, the marine sea bream accommodates calcium-transporting mechanisms to obtain calcium from the environment and preserve internal stores. These observations suggest that a fundamental difference may exist in the E2-dependent calcium regulation between freshwater and marine teleosts.     

Production and characterisation of gilthead sea bream (Sparus auratus) recombinant parathyroid hormone related protein

The production and purification of gilthead sea bream recombinant parathyroid hormone related protein [sbPTHrP(1-125)] using an Escherichia coli system and one step purification process with continuous elution gel electrophoresis is reported. The cDNA encoding sbPTHrP(1-125) was cloned into a prokaryotic expression vector pET-11a. The recombinant plasmid was used to transfect E. coli BL21(DE3) pLysS and sbPTHrP(1-125) synthesis was induced by addition of 1 mM isopropyl-β-d-thiogalactopyranoside. The rapid one step isolation method gave pure sbPTHrP(1-125) as judged by SDS-PAGE and yielded up to 40 mg/L of culture medium (3.3 mg protein/g of bacteria). The bioactivity of recombinant sbPTHrP(1-125) assessed using an in vitro scale bioassay was found to be equipotent to PTHrP(1-34) in stimulating cAMP accumulation. Assessment of the immunological reactivity of the isolated protein by Western blot revealed it cross-reacts with antisera specific for the N-terminal and C-terminal region of PTHrP. In a radioimmunoassay specific for piscine N-terminal (1-34 aa) PTHrP, the recombinant sbPTHrP(1-125) was equipotent with PTHrP(1-34) in displacing labelled ¹²⁵I-PTHrP(1-36) PTHrP from the antisera. The availability of recombinant sbPTHrP will allow the development of region specific assays and studies aimed at defining post-secretory processing of this protein and its biological activity in fish.     

Differential expression of PTHrP and its receptor in pituitary gland and gills in estradiol-treated gilthead sea bream (Sparus auratus, L.)

In the gilthead sea bream (Sparus auratus) 17β-estradiol (E₂) plays an important role in the synthesis of vitellogenin. During vitellogenesis, vitellogenin as a nutritional precursor protein is loaded with calcium, which requires elevated plasma calcium levels. This is accomplished via E₂-dependent processes. Reports have shown that hypercalcemic effects of E₂ are possibly mediated by another hypercalcemic factor, viz. parathyroid hormone related protein (PTHrP). To further investigate the possibility of PTHrP as a mediator of E₂-induced hypercalcemia, we investigated the local expression levels of the pthrp mRNA and of the gene coding for the PTHrP receptor, PTH1R (pth1r) in two tissues involved in the calcium regulation (gills, pituitary gland) of the sea bream treated with E₂. Compared to control, treatment with E₂ resulted in: significantly increased total calcium and plasma PTHrP levels (P < 0.01), a down-regulation of pthrp mRNA in the pituitary gland (P < 0.01), and up-regulation of expression levels for both pthrp and pth1r in the branchial system (P < 0.05). These findings provide direct evidence for a mediating role of PTHrP in E₂ induced hypercalcemia, and in addition support the idea for the presence of two independent systems, an endocrine pituitary PTHrP system and a peripheral paracrine branchial PTHrP system.     

Determination of tissue and plasma concentrations of PTHrP in fish: development and validation of a radioimmunoassay using a teleost 1-34 N-terminal peptide

A specific and sensitive radioimmunoassay (RIA) for the N-terminus of sea bream (Sparus auratus) and flounder (Platichthys flesus) parathyroid hormone-related protein (PTHrP) was developed. A (1-34) amino-terminal sequence of flounder PTHrP was synthesized commercially and used as the antigen to generate specific antiserum. The same sequence with an added tyrosine (1-35(Tyr)) was used for iodination. Human (1-34) parathyroid hormone (PTH), human (1-34) PTHrP, and rat (1-34) PTHrP did not cross-react with the antiserum or displace the teleost peptide. Measurement of PTHrP in fish plasma was only possible after denaturing by heat treatment due to endogenous plasma binding activity. The minimum detectable concentration of (1-34) PTHrP in the assay was 2.5 pg/tube. The level of immunoreactive (1-34) PTHrP in plasma was 5.2+/-0.44 ng/ml (mean+/-SEM, n=20) for flounder and 2.5+/-0.29 ng/ml (n=64) for sea bream. Dilution curves of denatured fish plasma were parallel to the assay standard curve, indicating that the activity in the samples was indistinguishable immunologically from (1-34) PTHrP. Immunoreactivity was present, in order of abundance, in extracts of pituitary, oesophagus, kidney, head kidney, gills, intestine, skin, muscle, and liver. The pituitary gland and oesophagus contained the most abundant levels of PTHrP, 37.7+/-6.1 ng/g wet tissue and 2.3+/-0.7 ng/g wet tissue, respectively. The results suggest that in fish PTHrP may act in a paracrine and/or autocrine manner but may also be a classical hormone with the pituitary gland as a potential major source of the protein.     

Parathyroid hormone-related protein and calcium regulation in vitamin D-deficient sea bream (Sparus auratus)

Gilthead sea bream (Sparus auratus L.) were fed a vitamin D-deficient diet for 22 weeks. Growth rate, whole body mineral pools and calcium balance were determined. Plasma parathyroid hormone-related protein (PTHrP) and calcitriol levels were assessed. Expression of mRNA for pthrp and pth1r was quantified in gills and hypophysis. Fish on vitamin D-deficient diet (D- fish) showed reduced growth and lower calcium turnover (calcium influx, efflux and accumulation rates decreased) and unaltered plasma calcium levels. Plasma calcitriol levels became undetectable, PTHrP levels decreased in the D- fish. In controls, a significant increase in plasma PTHrP level over time was seen, i.e. it increased with body mass. Relationships were found between plasma PTHrP and the whole body pools of calcium, phosphorus and magnesium, indicative of a role for PTHrP in bone development. Expression of pthrp and pth1r mRNA was down-regulated in the hypophysis of D-fish, whereas in gill tissue, pthrp and pth1r mRNA were up-regulated. We conclude that lower pthrp mRNA expression and plasma values in D- fish reflect lower turnover of PTHrP under conditions of hampered growth; up-regulation of pthrp mRNA in gills indicate compensatory paracrine activity of PTHrP during calcitriol deficiency to guarantee well-regulated branchial calcium uptake. This is the first report to document a relation between PTHrP and calcitriol in fish.     

Temporal expression of hepatic estrogen receptor 1, vitellogenin1 and vitellogenin2 in European silver eels

Because European silver eels have never been caught during or after their 6000-km reproductive migration to the Sargasso Sea, all existing knowledge on their sexual maturation comes from hormonal stimulation. Silver eels that start their oceanic migration are still immature with pre-vitellogenic oocytes. Hence we assumed that vitellogenesis should start with the expression of the estrogen receptor in the liver before the circulating 17β-estradiol (E2) can have any effect. In this study we followed the hepatic vitellogenesis upon 4 weekly injections with carp pituitary extracts (CPE). New molecular primers for the expression of the estrogen receptor 1 (esr1), vitellogenin1 (vtg1) and vitellogenin2 (vtg2) in the liver were developed. Sequences of vtg2 and esr1 were not previously described in Anguilla anguilla. All eels showed weekly increase of the eye size and pectoral fin length, which are signs of early maturation. The same occurred with the gonadosomatic index, the oocyte stage and diameter, and number of deposited fat droplets. Early vitellogenesis appeared as a 3-step process (1) E2-levels and esr1 expression were significantly increased already after one injection, (2) vtg1 and vtg2 expression were significantly increased after one and two injections, respectively, and (3) vtg1 and vtg2 expression increased further after three and four injections. Then also plasma calcium (corresponds with plasma vitellogenin) increased and yolk globuli appeared in the oocytes. These results show that esr1 is the first of the three genes examined that is expressed during the onset of hepatic vitellogenesis. Furthermore, ovarian vitellogenesis (appearance of yolk globuli in oocytes) occurs 1-2 weeks later than the onset of hepatic vitellogenesis.     

Melatonin synthesis under calcium constraint in gilthead sea bream (Sparus auratus L.)

Brain or blood plasma melatonin was analysed as a measure for pineal melatonin production in sea bream. Access to calcium was limited by diluting the seawater to 2.5 per thousand and removing calcium from the diet or by prolonged feeding of vitamin D-deficient diet. Interactions/relations between melatonin and calcium balance and the hypercalcemic endocrines PTHrP and calcitriol were assessed. Restricting calcium availability in both water and diet had no effect on plasma melatonin, but when calcium was low in the water or absent from food, increased and decreased plasma melatonin was observed, respectively. Fish on a vitamin D-deficient diet (D- fish) showed decreased plasma calcitriol levels and remained normocalcemic. Decreased brain melatonin was found at all sampling times (10-22 weeks) in the D- fish compared to the controls. A positive correlation between plasma Ca2+ and plasma melatonin was found (R(2)=0.19; N=41; P <0.01) and brain melatonin was negatively correlated with plasma PTHrP (R(2)=0.78; N=4; P <0.05). The positive correlation between plasma levels of melatonin and Ca2+ provides evidence that melatonin synthesis is influenced by plasma Ca2+. The decreased melatonin production in the D- fish points to direct or indirect involvement of calcitriol in melatonin synthesis by the pineal organ in teleosts. The hypercalcemic factors PTHrP and calcitriol appeared to be negatively correlated with melatonin and this substantiates an involvement of melatonin in modulating the endocrine response to cope with hypocalcemia. It further points to the importance of Ca2+ in melatonin physiology.     

Cortisol and parathyroid hormone-related peptide are reciprocally modulated by negative feedback

In previous in vitro studies, we have shown that the N-terminal region of parathyroid hormone-related protein (PTHrP) can stimulate cortisol production in sea bream, Sparus auratus, interrenal tissue, possibly through a paracrine action. In the current study, the systemic interaction between cortisol and PTHrP was studied in vivo. Sustained elevated blood cortisol levels, induced either by cortisol injection or confinement stress, suppressed circulating PTHrP 6 and 24-fold, respectively, by comparison to control fish. Dexamethasone treatment reduced cortisol levels, prevented the decrease of plasma PTHrP observed in confined fish and raised plasma PTHrP levels in non-confined fish. In contrast, a single injection of (1-34) PTHrP caused a short-term (within 30 min and up to 2.5 h) decrease in plasma cortisol. The antagonistic effects between PTHrP and cortisol were substantiated by an overall (data pooled from all experiments) highly significant negative correlation (r0=-0.745, p<0.001, n=115) between the plasma levels of the two hormones. Although the underlying mechanism of the interaction still has to be determined, the high levels of PTHrP in circulation and the existence of systemic regulation favour the hypothesis that in fish PTHrP may act as an endocrine factor, although the gland that produces it still remains to be identified.     

Influence of temperature regime on endocrine parameters and vitellogenesis during experimental maturation of European eel (Anguilla anguilla) females

We examined the effect of temperature in European silver eels during their maturation induced by injections of carp pituitary extract on endocrine parameters: pituitary fshβ and lhβ expression, plasma 17β-estradiol (E2) and vitellogenin, estrogen receptor 1 (esr1), and vitellogenin 2 (vtg2) expression in liver. A variable thermal regime (T10) that increased from 10° to 14° and 17 °C was compared with a constant 20 °C regime (T20) during 12 weeks. T10 caused a faster development until week 8, higher fshβ, lhβ, esr1 expression, and higher E2 levels. The results strongly suggest that T10 is inducing a higher endogenous FSH level which increases the E2 circulating level during vitellogenesis. A variable thermal regime induced an fshβ expression and E2 profile in vitellogenic hormonally matured eel females that were more similar to the profile observed in other naturally maturing fish.     

Vitellogenin synthesis via androgens in primary cultures of tilapia hepatocytes

Involvement of androgens in vitellogenin (VTG) synthesis was investigated using the primary hepatocyte cultures of tilapia, Oreochromis mossambicus. Concentration of VTG in the medium was assessed by enzyme-linked immunosorbent assay. When the hepatocytes of females were treated with testosterone (T), 17 alpha-methyltestosterone (MT) and 5 alpha-dihydrotestosterone (DHT), VTG concentration in the medium slightly increased or maintained. DHT, but not T and MT, increased VTG in the medium of male hepatocyte cultures. However, VTG production in the male hepatocytes, which were previously treated with estradiol-17 beta (E(2)), maintained high level by treatment of T. Similarly, co-treatment of E(2) and the androgens to the male hepatocytes enhanced VTG concentration in the medium. These results suggest that the androgens have some roles in VTG synthesis in the hepatocytes. Tamoxifen, a nonsteroidal antiestrogen, reduced VTG synthesis by the androgens. On the other hand, co-treatment of T and fadrozole, an aromatase inhibitor, failed to inhibit the effect of VTG synthesis by T alone. Analysis with RT-PCR did not demonstrate expression of the brain and the ovarian types of aromatase mRNA in the liver. These results suggest that the possibility of local aromatization of the androgens in the tilapia liver is low and that androgens bind estrogen receptor and, consequently, exert estrogenic action. Treatment of cyproterone acetate, an antiandrogen reagent, increased production of VTG with DHT. Involvement of androgens might not be ignored in regulation of VTG synthesis in the liver.     

Cloning of the cDNA for sea bream (Sparus aurata) parathyroid hormone-related protein

This paper reports cloning of the cDNA for sea bream (Sparus aurata) parathyroid hormone-related protein (PTHrP). The gene codes for a 125-amino acid mature protein with a 35-residue prepeptide. The total gene sequence is 1.8 kb with approximately 75% noncoding. The N-terminus of the protein resembles mammalian and chicken PTHrP peptides with 12 of the first 21 amino acids identical and for which there is homology with mammalian parathyroid hormone. Toward the C-terminus, the nuclear transporter region between residues 79 and 93 in sea bream is 73% homologous to tetrapod PTHrP, and the RNA binding domain, 96-117, is 50% homologous, moreover starting with the conserved lysine and terminating with the lysine/arginine sequence. Sea bream PTHrP differs significantly from mammalian and chicken PTHrP, having a novel 16-amino acid segment between residues 38 and 54 and completely lacking the terminal domain associated in mammals with inhibition of bone matrix lysis. RT-PCR and in situ hybridization of sea bream tissues show that the gene is expressed widely and the results confirm observations of a PTHrP-like factor in sea bream detected with antisera to human PTHrP.     

Stimulation of cortisol release by the N terminus of teleost parathyroid hormone-related protein in interrenal cells in vitro

The mode of action of PTHrP in the regulation of sea bream (Sparus auratus) interrenal cortisol production was studied in vitro using a dynamic superfusion system. Piscine (1-34)PTHrP (10(-6)-10(-11) M) stimulated cortisol production in a dose-dependent manner. The ED50 of (1-34)PTHrP was 2.8 times higher than that of (1-39)ACTH, and maximum increase in cortisol production in response to 10(-8) M of (1-34)PTHrP was approximately 7-fold lower than for 10(-8) M of (1-39)ACTH. In contrast to (1-34)PTHrP, piscine (10-20)PTHrP, (79-93)PTHrP, and (100-125)PTHrP (10(-9)-10(-7) M) did not stimulate cortisol production. The effect of piscine (1-34)PTHrP on cortisol production was abolished by N-terminal peptides in which the first amino acid (Ser) was absent and by simultaneous addition of inhibitors of the adenylyl cyclase-protein kinase A and phospholipase C-protein kinase C intracellular pathways but not by each separately. The PTHrP-induced signal transduction was further investigated by measurements of cAMP production and [H3]myo-inositol incorporation in an interrenal cell suspension. Piscine (1-34)PTHrP increased cAMP and total inositol phosphate accumulation, which is indicative that the mechanism of action of PTHrP in interrenal tissue involves the activation of both the adenylyl cyclase-cAMP and phospholipase C-inositol phosphate signaling pathways. These results, together with the expression of mRNA for PTHrP and for PTH receptor (PTHR) type 1 and PTHR type 3 receptors in sea bream interrenal tissue, suggest a specific paracrine or autocrine steroidogenic action of PTHrP mediated by the PTHRs.     

CYP27A1 expression in gilthead sea bream (Sparus auratus, L.): effects of calcitriol and parathyroid hormone-related protein

Little is known about vitamin D metabolism in fishes. Several reports have shown hydroxylase activities in various organs to produce vitamin D metabolites, but the enzymes involved have not been isolated or characterized. We isolated and characterized a renal mitochondrial hydroxylase, CYP27A1, that governs vitamin D metabolism in gilthead sea bream, Sparus auratus. The enzyme is highly expressed in kidney and to a far lesser extent in liver. When treated with 25-hydroxy vitamin D or calcitriol, the kidney responded differentially and time dependently with CYP27A1 mRNA expression levels. This response substantiates a role for CYP27A1 in fish vitamin D metabolism. This notion is strengthened by upregulation of CYP27A1 in sea bream treated with parathyroid hormone-related protein (PTHrP), and suggests an original role for PTHrP in calcitriol-regulated processes n fish similar to the role of PTH in mammalian vitamin D-dependent processes.     

Lipids and lipid-transporting proteins in Chrysemys picta: role of gonadal steroids and growth hormone in intact and hypophysectomized turtles

In the freshwater turtle, the homeostatic control of plasma lipids and lipid-transporting proteins may be coordinately regulated by ovarian steroids and pituitary hormones such as growth hormone (GH). In order to elucidate the role of these hormones in the regulation of vitellogenesis and ovarian growth, we have investigated lipid metabolic changes in normal male and female turtles, and in hypophysectomized females with and without GH injections, in response to combinations of exogenously administered gonadal steroids (estradiol (E2), progesterone (P), and testosterone (T)). Determinations of total plasma triglycerides, cholesterol, vitellogenin, and apoA-I were performed. We have demonstrated that E2 alone and in combination with P significantly increased plasma apoA-I and triglyceride levels in both intact female and male turtles. Testosterone administered alone to males had no effect on any of the parameters measured. In hypophysectomized females, plasma apoA-I, vitellogenin, and triglyceride levels were all significantly elevated in animals which received GH and E2, compared to controls (sham and hypox) and those which received GH alone.     

Differences in binding characteristics of sex steroid binding protein in reproductive and nonreproductive female rainbow trout (Oncorhynchus mykiss), black bream (Acanthopagrus butcheri), and greenback flounder (Rhombosolea tapirina)

The binding characteristics of sex steroid binding protein (SBP) were investigated in vitellogenic and nonreproductive female rainbow trout (Oncorhynchus mykiss), black bream (Acanthopagrus butcheri), and greenback flounder (Rhombosolea tapirina). The binding capacity of rainbow trout and black bream SBP was significantly greater in vitellogenic than in nonreproductive-stage fish. A decrease in binding affinity was observed in male trout injected with estradiol (E(2)) compared to control fish. This difference was not observed after partial purification of the SBP by gel filtration and may have resulted from competitive inhibition of E(2) binding by vitellogenin. No differences in flounder SBP binding characteristics were observed.     

The regulatory action of estrogen and vasoactive intestinal peptide on prolactin secretion in sea bream (Sparus aurata,L.)

The effect of estradiol-17beta (E(2)) implants on the in vitro secretion of prolactin (PRL) and its modulation by vasoactive intestinal peptide (VIP) in a marine teleost, sea bream (Sparus aurata L.), was determined. Experiments were conducted during winter and spring. During winter, fish (n=130, body weight 50-70 g) were randomly divided into 2 groups; control and E(2) treated (10 mg/kg, wet weight). Fish were sacrificed after 7 days treatment and in vitro pituitary cultures in Ringer bicarbonate supplemented with increasing doses (0-200 nM) of VIP were carried out for 18 h. Culture medium was analysed by PAGE and secreted PRL quantified by densitometry. Fish treated with E(2) secreted significantly more PRL (P<0.05) in vitro than control fish. In E(2) primed fish VIP caused a dose-dependent inhibition of PRL secretion in vitro. VIP had no detectable effect on the secretion of PRL from control pituitaries. Treatment with E(2) had a different effect during spring; PRL secretion was significantly decreased (P<0.01) compared with the control fish. Anatomical evidence of abundant VIP immunoreactive nerve fibres in neurohypophysial (NH) tissue penetrating the rostral pars distalis provide further evidence supporting an action for VIP in the regulation of PRL cells. In conclusion, the responsiveness of PRL in the pituitary gland varied with season. Moreover, in the sea bream VIP appears to modulate PRL secretion from E(2) primed pituitary glands.